The phenomenon of light emission by living organisms, known as bioluminescence, is a characteristic of different species including, for example, insects and bacteria. Most of the world's luminescent bacteria are represented by marine species, including Vibrio, Photobacterium, Photorhabdus and Shewanella families. Some of these organisms live as free water-born bacteria, while others as symbionts in light organs of marine animals.
Non-bacterial organisms such as plants that are capable of bioluminescence would be useful for many purposes, such as for environmental and aesthetic applications. However, such organisms have not been readily achieved for many reasons. For example, the genes and mechanisms responsible for bioluminescence are complex.
Genetic engineering of plants has been typically limited to introduction of one or two new genes into plant genome. This limitation prevents incorporation of complex metabolic pathways, such as those involved in light emission, into transgenic plant organisms.
Conventionally constructed genetic transformation vectors are made using “regular” restriction endonucleases with average recognition site length of six nucleotides. Such construction is a laborious and time consuming process, which involves sequential cloning of different functional plasmid elements (e.g., promoters, terminators, and integration sequences) by a series of multiple cloning steps.
After cloning several vector segments, commonly used restriction endonucleases sites will be present in the newly inserted DNA fragments (due to high statistical probability of occurrence of a six base pair sequence in an extended DNA fragment). The presence of such sites significantly limits the number and size of additional DNA sequences that may need to be added to achieve a desired biological property such as bioluminescence. Moreover, if initially cloned DNA elements are required to be exchanged to yield another property, the entire vector typically must be reconstructed. It is often impossible to remove previously cloned genetic elements without affecting later cloned sequences.
Incorporation of multiple transgenes into a single plant organism has been attempted using standard breeding techniques. However, such an approach is time consuming and largely ineffective. Accordingly, a genetic vectors system allowing for efficient incorporation of multiple transgenes is needed. An approach for allowing rapid exchange of multiple transgenes in the genome of any selected plant species (especially the plastid genome) is also needed.